The general goal of this project is to determine the dynamic[unreadable] organization and regulation of specific cytoskeletal components in[unreadable] living cells, using a combination of chemical probes, imaging[unreadable] techniques, and biophysical analyses. Experiments in the next funding[unreadable] period will start with the development of three new probes that should[unreadable] expand our abilities to study molecular functions in vivo: a probe that[unreadable] reveals cell adhesion forces, a probe that depolymerizes actin filaments[unreadable] upon photoactivation, and a probe that facilitates the detection of[unreadable] single molecules. These probes will then be applied in conjunction with[unreadable] existing methods including fluorescent analog cytochemistry, computer[unreadable] image restoration, photobleaching, and resonance energy transfer imaging[unreadable] to study molecular interactions during cell locomotion, cell-substrate[unreadable] adhesion, and cytokinesis. Experiments will be performed to determine[unreadable] the distribution and magnitude of traction forces under moving[unreadable] fibroblasts, the corresponding organization of contractile structures,[unreadable] and the regulatory roles of protein phosphorylation and microtubules.[unreadable] In addition, the assembly of actin will be studied at focal adhesions,[unreadable] where it might play a role in maintaining structural integrity and in[unreadable] force generation. The dynamics of vinculin will be examined in relation[unreadable] to a putative regulatory mechanism involving conformational changes.[unreadable] Experiments will also be performed to probe the mechanism of[unreadable] cytokinesis, by mapping the contribution of different regions of the[unreadable] cortex in generating cleavage forces, and by determining the spatial[unreadable] relationship between microtubles and the cortex. These experiments[unreadable] should provide crucial basic knowledge for understanding not only normal[unreadable] physiological functions, such as embryonic development and wound[unreadable] healing, but also a number of diseases related to cell locomotion or[unreadable] division including birth defects and cancer.